U.S. patent number 4,800,078 [Application Number 07/055,008] was granted by the patent office on 1989-01-24 for immunotherapeutic method of treating respiratory disease by intranasal administration of igb.
This patent grant is currently assigned to The United States of America as represented by the Secretary of the. Invention is credited to Robert Chanock, Val G. Hemming, Gregory Prince.
United States Patent |
4,800,078 |
Prince , et al. |
January 24, 1989 |
Immunotherapeutic method of treating respiratory disease by
intranasal administration of Igb
Abstract
A new immunotherapeutic method of treating lower respiratory
tract infection caused by respiratory syncytial virus (RSV) is
disclosed. The method employs topical application of RSV antibodies
into the lower respiratory tract. The new treatment modality is
more effective and rapid than the conventional therapy.
Inventors: |
Prince; Gregory (Bethesda,
MD), Chanock; Robert (Bethesda, MD), Hemming; Val G.
(Gaithersburg, MD) |
Assignee: |
The United States of America as
represented by the Secretary of the (Washington, DC)
|
Family
ID: |
21994974 |
Appl.
No.: |
07/055,008 |
Filed: |
May 28, 1987 |
Current U.S.
Class: |
424/159.1;
424/177.1 |
Current CPC
Class: |
A61K
9/0043 (20130101); A61K 9/0073 (20130101); C07K
16/1027 (20130101) |
Current International
Class: |
A61K
9/00 (20060101); C07K 16/10 (20060101); C07K
16/08 (20060101); A61K 039/42 (); A61K
039/395 () |
Field of
Search: |
;530/387
;514/956,957,958,997 ;424/86,85 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
Other References
Prince et al., J. Virology, 55:517-520, 1985. .
Hemming et al., J. Infectious Dis., 152:1083-1087, 1985. .
Prince et al., Virus Research, 3:193-206, 1985. .
Prince et al., Pediatric Infectious Disease, 5:S201-S203, 1986.
.
Annals of the New York Academ of Science, vol. 443:67-68, 1985,
Edited by Norman S. Braveman et al. .
Rabin et al., Pav. J. Biol. Sci., 1986, vol. 21, No. 2..
|
Primary Examiner: Schain; Howard E.
Assistant Examiner: Kushan; Jeff
Attorney, Agent or Firm: Holman & Stern
Claims
What is claimed is:
1. A method of treating respiratory disease caused by respiratory
scintial virus (RSV), consisting essentially of administering into
the lower respiratory tract of a host suffering from respiratory
scintial disease of the lower respiratory tract, a single dose of
about 0.025 to about 0.05 g/Kg body weight of purified human gamma
globulin suitable for administration by intravenous route and
containing respiratory scintial virus-neutralizing antibodies at a
titer greater than 1:2000 to produce therapeutic effect against
disease caused by RSV.
Description
BACKGROUND OF THE INVENTION
1. Technical Field
The present invention is related to immunotherapy of respiratory
disease caused by respiratory syncytial virus (RSV) or other
respiratory viruses. More particularly, the present invention is
related to a more effective and rapid method of treating lower
respiratory tract disease caused by RSV by direct administration of
neutralizing antibodies into the lower respiratory tract.
2. State of the Art
Lower respiratory tract infection caused by RSV is a serious
problem in infants and children, particularly infants under six
months of age. Currently licensed therapy for RSV requires the
delivery of ribavirin
(1-beta-D-ribofuranosyl-1,2,4,-triazole-3-carboxamide) by small
particle aerosol for 12-20 hours a day for at least 3 days (Hall et
al, 1983 New Eng. J. Med. 308: 1443-1447; Taber et al, 1983
Pediatrics 72: 613-618). A more effective and quick acting
treatment of the respiratory disease caused by RSV is, therefore,
an obvious need.
SUMMARY OF THE INVENTION
It is, therefore, an object of the present invention to provide a
more efficacious, simpler and quick acting method of treating
virus-caused respiratory disease than the currently available
therapeutic modalities.
Other objects and advantages will become evident from the Detailed
Description of the Invention.
DETAILED DESCRIPTION OF THE INVENTION
The above and various other objects and advantages of the present
invention are achieved by a method of treating respiratory disease,
comprising topically administering to a host susceptible to or
suffering from infection by respiratory syncytial virus (RSV), an
effective amount of neutralizing antibodies to produce prophylactic
or therapeutic effect against virus infection.
Unless defined otherwise, all technical and scientific terms used
herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
any methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, the preferred methods and materials are now described.
All publications mentioned hereunder are incorporated herein by
reference.
MATERIALS AND METHODS
Animals
Cotton rats (Sigmodon hispidus) and owl monkeys were obtained from
the Veterinary Resources Branch, Division of Research Services,
National Institues of Health. A small nucleus colony of cotton
rats, maintained behind a germ-free barrier for the past 10 years,
provided animals for the production colony. Adult cotton rats were
immunized with inactivated Sendai virus vaccine (Microbiological
Associates) at least 3 weeks before study. Sigmodon fulviventer
were obtained from the same source, initiated from wild-trapped
animals.
IVIG Administration
A single lot of purified human IVIG (Sandoglobulin, lot
#2.370.069.0) was used for all experiments. This lot had a RSV
neutralizing antibody titer of 1:2905. Cotton rats infected three
days earlier by intranasal instillation of A2 strain of RSV
(10.sup.4 pfu/animal) were anesthetized with methoxyflurane and
inoculated intraperitoneally (IP) or intranasally (IN) with a
varying dose of IVIG. The volume of IVIG inoculated IN was 0.1
ml/100 gm body weight. Intranasal administration of IVIG to fully
anesthetized cotton rats or monkeys resulted in rapid delivery of
the inoculum into the lungs.
Virus Assay
Cotton rats were sacrificed by carbon dioxide asphyxiation 24 hours
after administration of IVIG. Lungs and nasal tissues (including
nasal turbinates) were homogenized in 10 parts (w/v) of Hanks'
balanced salt solution supplemented with 0.218M sucrose, 4.4 mM
glutamate, 3.8 mM KH.sub.2 PO.sub.4 and 3.2 mM K.sub.2 HPO.sub.4,
and the resulting suspension was stored at -70 degrees C. until
assayed. Virus titer was determined by plaque assay on HEp-2 cell
monolayers as described by Prince et al (1978) Am. J. Pathol. 93:
771-792, and was expressed in plaque-forming units (pfu)/gm
tissue.
Viral assay in owl monkeys was similarly conducted using tracheal
lavage from the animals.
Antibody and Immunoglobulin Assays
Neutralizing antibody was measured by a plaque-reduction
neutralization assay as described by Prince et al, supra, using a
60% plaque-reduction endpoint. Human IgG in cotton rat serum was
measured by radial immunodiffusion (Meloy Laboratories,
Springfield, Va.) and low levels (<10 mg/dl) were confirmed by
standard competitive inhibition ELISA such as described by Boswirth
et al, 1983, J. Immunol. Methods 62: 331-336.
Histology
Lungs were removed from the thorax and inflated through the trachea
with neutral buffered formalin. Histologic sections were stained
with hematoxylin and eosin.
Comparison of therapeutic efficacy of IVIG administered
parenterally (IP) or topically (IN)
Administration of IVIG by the IP route, in a dose of 2 gm/kg or
more, 3 days after infection of the respiratory tract with RSV,
reduced the titer of RSV in the lungs 10.sup.-2 or more within 24
hours. As a consequence virus could not be detected on the fourth
day post-infection in pulmonary tissue of 80-90% of animals treated
in this manner (Table 1). A significant reduction in the quantity
of virus in the nose was also observed, but this effect was not as
dramatic as that observed in the lungs.
IVIG administered by the intranasal route is more effective than
IVIG inoculated parenterally. Thus, topically administered IVIG
cleared pulmonary RSV in every animal when a dose of 0.05 gm/kg or
greater was used. However, IVIG administered in this way had no
effect on the quantity of RSV present in the nose (Table 1). The
therapeutic effect of IN IVIG on pulmonary viral titer diminished
with decreasing dose and the minimum effective does was observed to
be 0.00625 gm/kg (p<0.001). Cotton rats inoculated topically
with the largest dose of IVIG (0.1 gm/kg), attained near peak
levels of this material in serum within 5 hours, but RSV antibodies
did not reach a concentration which allowed their detection by the
neutralization assay. The concentration of IVIG in serum 24 hours
after topical administration ranged from 61 to 140 .mu.g/ml for
cotton rats which received 0.025 to 0.05 gm/Kg body weight, a dose
which cleared or substantially (almost completely) cleared
pulmonary RSV. The concentration of IVIG in the serum of cotton
rats which exhibited a similar therapeutic effect when IVIG was
administered parenterally (4 to 8 gm/Kg body weight) was 120 to 202
times higher, i.e., 12,379 to 16,829 .mu.g/ml (Table 1).
TABLE 1
__________________________________________________________________________
Therapeutic Effect of Human IVIG Administered Intraperitoneally
(IP) or Intranasally (IN) Three Days Post Infection Level in serum
one day following IVIG treatment Viral titer 4 days post-infection,
Route IVIG administered Dose IVIG Titer of RSV log.sub.10 pfu/gm
geometric mean .+-. S.E. 3 days post-infection (gm/kg body # of
neutralizing antibodies (% of animals free of detectable virus)
(10.sup.4.0 pfu RSV) weight) animals (reciprocal) IVIG (.mu.g/ml)
Lungs Nose
__________________________________________________________________________
Control -- 24 <20 0 4.26 .+-. 0.08 (0) .sup. 4.80 .+-. 0.17
(0).sup. I.P. 8.0 13 692 16,829 2.15 .+-. 0.10.sup.a 2.82 .+-.
0.36.sup.a (62) 4.0 10 518 12,379 2.04 .+-. 0.05.sup.a 3.57 .+-.
0.36.sup.b (10) 2.0 10 302 4,769 2.26 .+-. 0.19.sup.a 3.61 .+-.
0.44.sup.b (20) 1.0 8 142 3,760 3.27 .+-. 0.27.sup.a 4.85 .+-.
0.14.sup.c (0) 0.25 4 59 1,670 4.13 .+-. 0.03.sup.c (0) 4.81 .+-.
0.07.sup.c (0) I.N. 0.1 13 <20 200 <2.0.sup.a (100) 4.96 .+-.
0.14.sup.c (0) 0.05 11 <20 140 <2.0.sup.a (100) 4.74 .+-.
0.21.sup.c (0) 0.025 12 <20 61 2.06 .+-. 0.06.sup.a 4.85 .+-.
0.15.sup.c (0) 0.0125 10 NT NT 2.78 .+-. 0.19.sup.a 4.36 .+-.
0.26.sup.c (0) 0.00625 10 NT NT 3.50 .+-. 0.14.sup.a (0) 4.30 .+-.
0.25.sup.c (0) 0.003125 10 NT NT 4.15 .+-. 0.14.sup.c (0) 4.86 .+-.
0.27.sup.c (0) 0.0015625 12 NT NT 4.86 .+-. 0.12.sup.c (0) 5.39
.+-. 0.17.sup.c
__________________________________________________________________________
(0) Significance of reduction of viral titer compared to infected
animals not treated IVIG: .sup.a p < 0.001 .sup.b p < 0.005
.sup.c not significant NT = Not tested
Therapeutic effect of IN IVIG not due to neutralization in
vitro
The possibility that the observed reduction in pulmonary viral
titer of animals treated by topical administration of IVIG could be
due to neutralization in vitro was evaluated by mixing an equal
amount of infected lung tissue from a treated cotton rat (human
IVIG administered I.N. at a dose of 0.1 gm/kg 3 days post
infection) with infected lung tissue from a cotton rat which had
not received IVIG. Mixed homogenates yielded the same amount of
virus as lung suspensions from infected cotton rats that had not
received IVIG (Table 2). These results indicate that the
therapeutic effect observed in IVIG recipients was due to bona fide
passive immunity rather than neutralization of virus in vitro
following homogenization of lung tissue.
TABLE 2 ______________________________________ Therapeutic Effect
of IVIG Administered Topically Post Infection Not Attributable to
In Vitro Neutralization During Homogenization of Tissues
RSV-infected No. of Titer of virus in lungs 4 days after lung
tissue cotton challenge with 10.sup.4 pfu of RSV homogenized rats
(geometric mean, log.sub.10 pfu/gm .+-. S.E.)
______________________________________ Group ARSV
antiserumadministered 3days after virusGroup BAntiserum
notadministeredGroup CMixture ofgroup A and Btissues 444 + 4
##STR1## ______________________________________ .sup.a P < 0.001
.sup.b Not significant
Topical IVIG therapy does not prolong infection
The possibility was also examined that topical administration of
IVIG (0.1 gm/kg) might reduce viral titer on the 4th day
post-challenge, but prolong the course of infection. This question
was addressed by comparing the titer of RSV in tissues of infected
IVIG-treated and control animals at various intervals after
treatment (Table 3). In each instance tissues from IVIG-treated
animals yielded significantly smaller amounts of virus than tissues
from control animals and there was no evidence of a rebound in
virus replication.
TABLE 3
__________________________________________________________________________
Reduction In Level of Pulmonary RSV by Topical Administration of
IVIG Is Not Followed By Rebound Pulmonary viral titer (log.sub.10
pfu/gm geometric mean .+-. S.E.) at intervals (days) Species of
after IVIG (0.1 gm/kg) was administered I.N. on third day
post-infection cotton rat 1 2 3 4 5 7
__________________________________________________________________________
S. hispidus IVIG- <2.0 NT 2.41 .+-. 0.32 NT NT <2.0 treated
.sup. (4).sup.a (4) (4) Untreated 4.69 .+-. 0.30 NT 4.57 .+-. 0.16
NT NT <2.0 (4) (4) (4) S. fulviventer IVIG- 2.61 .+-. 0.62 2.73
.+-. 0.63 2.61 .+-. 0.42 <2.0 <2.0 <2.0 treated (4) (4)
(4) (4) (4) (5) Untreated 5.05 .+-. 0.13 4.96 .+-. 0.1 3.75 .+-.
0.19 3.05 .+-. 0.36 <2.0 <2.0 (4) (4) (4) (4) (4) (2)
__________________________________________________________________________
N.T. = not tested .sup.a number of animals
Topical IVIG not associated with increased pulmonary
immunopathology
It has been reported that cotton rats previously vaccinated with
formalin-inactivated RSV developed enhanced pulmonary pathology,
resembling an Arthus reaction, when they were infected with RSV
(Prince et al, 1986, J. Virol. 57: 721-728). In order to determine
whether immunotherapy might initiate immunopathology, lung tissue
was harvested from infected cotton rats treated IN with IVIG (0.1
gm/kg) on the third day post-infection and the material examined
histologically. Tissues taken 24 and 96 hours after IN
administration of IVIG showed no evidence of pathology.
Prophylactic effect of topical IVIG
The possibility that topically-administered IVIG might prove useful
during seasonal or situational RSV epidemics for short-term
prophylaxis in high-risk situations (such as infants in an
intensive-care nursery or patients with congenital heart or
pulmonary disease) was examined by administering IVIG IN (0.1
gm/kg) prior to infection and then challenging animals with RSV IN
(10.sup.4 pfu/animal) at intervals up to seven days later. Four
days after challenge, the animals were sacrificed and the titer of
pulmonary virus was assayed (Table 4). Although the degree of
protection was inversely proportional to the interval between IVIG
inoculation and viral challenge, significant protection
(p<0.005) was detected as long as 7 days after the
immunoglobulin preparation was administered.
TABLE 4
__________________________________________________________________________
Topically-Administered IVIG Retains Antiviral Activity in the Lungs
for Up to Seven Days Pulmonary viral titer (log.sub.10 pfu/gm
geometric mean .+-. S.E.), 4 days post infection, of cotton rats
administered IVIG IN at Species of indicated interval prior to
infection (10.sup.4 pfu of RSV IN) cotton rat Untreated 1 hour 1
day 2 days 4 days 7 days
__________________________________________________________________________
S. hispidus 5.85 .+-. 0.10 <2.0.sup.a 3.24 .+-. 0.43.sup.a 4.26
.+-. 0.43.sup.a 4.99 .+-. 0.06.sup.a 5.39 .+-. 0.09.sup.b (8)* (4)
(5) (5) (5) (6) S. fulviventer 5.83 .+-. 0.17 2.05 .+-. 0.06.sup.a
3.32 .+-. 0.33.sup.a 3.29 .+-. 0.51.sup.a 3.40 .+-. 0.33.sup.a 4.73
.+-. 0.21.sup.b (9) (5) (6) (5) (9) (7)
__________________________________________________________________________
Significance of reduction of viral titer compared to infected
animals not treated with IVIG: .sup.a p < 0.001 .sup.b p <
0.005 *Number of animals
Efficacy of Topical administration in owl monkeys
The efficacy of topically-administered antibody in owl monkeys is
shown in Table 5.
TABLE 5 ______________________________________ EFFICACY OF
TOPICALLY-ADMINISTERED ANTIBODY IN OWL MONKEYS PREVIOUSLY INFECTED
WITH RESPIRATORY SYNCYTIAL VIRUS Pulmonary viral titers Treatment
at (log.sub.10 pfu/m.l., .+-. S.E.) Day 5 No. of Day 5
Post-infection Animals (Pre-treatment) Day 7
______________________________________ 400 mg/Kg IgG 10 4.26 .+-.
0.10 1.53 .+-.0.26* Intranasally Untreated 6 4.30 .+-. 0.34 3.56
.+-. 0.09* ______________________________________ *p 0.001
In summary, the comparative study of the therapeutic efficacy of
IVIG administered topically or parenterally described herein shows
that whereas 0.025 gm/Kg of IVIG administered topically by the
intranasal route effected a 10.sup.2.2 -fold reduction in pulmonary
viral titer and complete clearance of detectable virus in 92% of
animals, 4 gm/Kg of IVIG was required to produce a comparable
therapeutic effect when the material was administered parenterally.
Thus, the therapeutic effect of IVIG was 160 times greater by the
topical route than by parenteral inoculation. For usage in humans,
topical therapy can be accomplished by administering the
immunoglobulin in such forms as small particle aerosol or the
like.
The results further indicate that therapeutic effect of IVIG is
permanent because rebound of virus replication in the lungs was not
detected. Moreover, as contrasted with previous studies with
formalin-treated vaccine, histologic evidence of immunopathology
was not seen in the lungs of infected animals which were treated
with IVIG topically.
Without being bound to any specific theory, it is postulated that
the therapeutic effect of topically administered IVIG appears to be
mediated within the lumen of the lung rather than within its
parenchyma. The quantity of IVIG present in the serum of topically
treated cotton rats which received the smallest dose capable of
almost completely clearing RSV from the lungs (i.e., 0.025. gm/Kg)
was 61 .mu.g/ml. This level was 200 times lower than the 12,379
.mu.g/ml of IVIG which was present in the serum of animals treated
parenterally with the minimum dose that almost completely cleared
RSV from the lungs, (i.e., 4 gms/Kg). Thus, the amount of IVIG in
the serum of infected cotton rats successfully treated topically
with immunoglobulin was substantially below the concentration of
IVIG associated with a full therapeutic effect in parenterally
treated animals. This implies that IVIG exerts its therapeutic
effect within the air passages of the lungs.
The surprising finding to emerge from this study was the duration
of activity of topically administered antibodies, wherein a single
IN inoculation of IVIG provided significant resistance for seven
days. For protecting children or infants, the RSV antibodies are
administered by aerosol means or the like to high-risk infants
hospitalized during RSV epidemics, no more than about twice a week,
thereby substantially reducing the risk of nosocomial infection and
disease. A single dose of IVIG, rapidly delivered by small particle
aerosol for therapy in serious RSV lower respiratory tract disease
in young infants now also becomes possible. It should be noted that
compared to the quick results obtained by the topical application
as disclosed by the present invention, the currently available
therapy for RSV requires the delivery of ribavirin by small
particle aerosol device for 12-20 hours a day for at least 3
days.
Clearly, therefore, the present invention opens a new vista for
more effective and rapid prevention and treatment of lower
respiratory tract viral infection and disease through the topical
administration of virus-specific antibodies. A definitive advantage
of the new treatment modality disclosed herein is that a material
which is already licensed for parenteral use in humans can now also
be applied via topical route for better results. The same strategy
could be utilized to treat or prevent severe, acute lower
respiratory tract disease caused by other important viral
respiratory pathogens such as influenza A, influenza B,
parainfluenza types 1, 2 and 3, and adenovirus.
It is understood that the examples and embodiments described herein
are for illustrative purposes only and that various modifications
or changes in light thereof will be suggested to persons skilled in
the art and are to be included within the spirit and purview of
this application and scope of the appended claims.
* * * * *